The primary aim of this project is to gain a better understanding of the mechanism by which neurophysins are synthesized by the neurons in the hypothalamus. Major efforts will be directed to the studies that can provide direct information in answering the questions of whether neurophysins are synthesized through larger precursors and if so whether neurophysins and their respective neurohormones, oxytocin and vasopressin share common precursors as has been suggested. The immediate goal of this proposal is to obtain sufficient quantities of radiolabeled putative precursors for further characterization. To achieve this goal, a cell-free protein synthesizing system from wheat germ extract will be used to translate messenger RNA's isolated from hypothalamic tissue. Phenol-chloroform-sodium dodecyl sulfate extraction followed by affinity chromatography on oligo-(dT) cellulose column will be used to isolate poly (A)-containing RNAs. Further fractionation of RNAs according to their molecular sizes will be carried out by column chromatography on Sepharose 4B and by centrifugation in sucrose density gradients. The translation products will be starting materials for the isolation of putative precursors. Rabbit antisera against procine neurophysins are used to precipitate proteins that are structurally related to porcine neurophysins. Electrophoresis of immunoprecipitates on polyacrylamide gels containing sodium dodecly sulfate and urea will be used to facilitate the determination of molecular weights and also the purification of putative precursors. Further characterization of the purified precursors will include enzymatic cleavage and peptide mapping. This proposal seeks answers to a long standing question concering the biosynthesis of neurophysins and neurohormones by using a new approach. An unequivocal solution to the question should constitute a significant advance in our knowledge on the mechanism of the biosynthesis of neurosecretory proteins and peptides of the hypothalamus.